The Role And Mechanism Of ASRGL1 In The Proliferation And Migration Of Hepatocellular Carcinoma

Objective: Hepatocellular carcinoma(HCC)is a disease with high morbidity and mortality,which seriously endangering human health.Despite of the progress have been made in HCC diagnosis and treatment,the postoperative recurrence rate still remains high,the prognosis the HCC patients remain poor,and there is a lack of effective diagnostic and therapeutic targets.Therefore,it’s of great significance to effectively improve the clinical diagnosis and treatment of HCC by further exploring the specific mechanism of HCC metastasis and recurrence.With the rapid development of transcriptomics,many studies have identified key molecules which might regulate recurrence and metastasis in HCC,and play an important role in the occurrence and recurrence of HCC.We previously identified ASRGL1 as a recurrence-related gene via high-throughput microarray in HCC,and validated the expression and prognostic value in public databases.Moreover,we explored the function and mechanism via in vivo and in vitro experiments,which might provide a theoretical basis for ASRGL1 as a therapeutic target for HCC metastasis and recurrence.Methods: 1.Four cases of HCC samples recurrence within 1-2 years after surgery(recurrence prone group)and non-recurrence samples within5 years after surgery(non-recurrence prone group)were collected in our hospital to construct a high-throughput microarray for sequencing,and the differentially expressed candidate genes were obtained through the analysis of the early recurrence group and the non-recurrence group.Analysis was performed to verify its differential expression in tumor and adjacent tumor tissue.Combined with clinical case information for correlation analysis,the Kaplan-Meier method was used to analyze the relationship with prognosis(disease-free survival and overall survival).2.We collected 12 pairs of HCC and its corresponding adjacent tissues,and extracted tissue RNA by Trizol method,amplify the target gene by ordinary PCR,and verify the transcription level by nucleic acid agarose gel electrophoresis.Next,we used immunohistochemistry and Western blot to verify the expression of ASRGL1 in HCC and its cancer.3.Quantitative real-time PCR(q RT-PCR)was used to detect the expression levels of target genes in seven HCC cell lines.4.Screening of relatively low-expressing cell lines in the above cell lines to construct stable over-expressing cell lines.Knock down the target gene in relatively high-expressing cell lines by transfecting si RNA.The effect of target gene on proliferation and migration was verified by plate cloning,CCK-8 and Transwell respectively;the effect of target gene on apoptosis was detected by flow cytometry.5.In vivo,we used mouse subcutaneous tumor model to verify the effect of target gene on tumorigenic ability and tumor biological behavior.6.RNA-seq was used to sequence the target gene overexpression and control cell lines.Next,we analyzed the differentially expressed genes and perform GO,KEGG and GSEA enrichment analysis to explore their downstream impact pathways.7.Validation of downstream up-regulated genes suggested by RNA-seq using q RT-PCR and flow cytometry.8.Western Blot was used to validate downstream potential pathways suggested by enrichment analysis.9.CIBERSORT was performed to analyze the effect of ASRGL1 on immune cell infiltration.10.We constructed and verified the mouse liver cancer cell line Hepa1-6which stably overexpressing Asrgl1,and constructed a subcutaneous tumor model to verify the level of immune cell infiltration.Results: 1.High-throughput microarray results suggested that ASRGL1 was one of the most differentially expressed candidate genes in the recurrence-prone and non-recurrence-prone groups.TCGA,OEP000321 and clinical sample analysis showed that ASRGL1 was significantly overexpressed in HCC tissues.It was highly expressed in Huh7 cell line,but relatively low in Bel-7405 cell line.2.In vitro,when ASRGL1 was knocked down,it was found that the proliferation and migration of HCC cells were weakened;Consistently,the proliferation,migration and anti-apoptosis abilities of HCC cells were enhanced after the overexpression of ASRGL1.It was found that high ASRGL1 promotes tumor growth in nude mice.3.RNA-seq results showed that CD36 was a potential downstream gene of ASRGL1,which was verified by q RT-PCR and flow cytometry,respectively.4.ASRGL1 might be associated with the ECM-receptor interaction pathway and may affect the progression of liver cancer through epithelialmesenchymal transformation(EMT).5.The subcutaneous tumor model of nude mice was constructed by using Bel-7405 stable overexpression cell line.The results showed that the tumor growth rate and tumor volume in the overexpression ASRGL1 group were significantly greater than those in the control group.6.In addition,CIBERSORT showed that ASRGL1 was positively correlated with the infiltration level of macrophages,and animal experimental results also suggested that highly expressed ASRGL1 could recruit macrophages in mouse tumors and peripheral blood.Conclusions: 1.ASRGL1 could promote the proliferation,migration and apoptosis of HCC cells,and affect the EMT of HCC cells through CD36.2.ASRGL1 could recruit macrophages and influence the immune microenvironment of HCC,thus promoting the progression of HCC.